Condensed pyrazindione derivatives

ABSTRACT

Compounds of the general structural formula (I) and use of the compounds and salts and solvates thereof, as therapeutic agents.

CROSS REFERENCE TO RELATED APPLICATION

This is the U.S. national phase application of International ApplicationNo. PCT/US01/15550, filed May 15, 2001, which claims the benefit of U.S.provisional patent application Ser. No. 60/214,284, filed Jun. 26, 2000.

FIELD AND BACKGROUND OF THE INVENTION

This invention relates to a series of compounds, to methods of preparingthe compounds, to pharmaceutical compositions containing the compounds,and to their use as therapeutic agents. In particular, the inventionrelates to compounds that are potent and selective inhibitors of cyclicguanosine 3′,5′-monophosphate specific phosphodiesterase (cGMP-specificPDE), in particular PDE5, and have utility in a variety of therapeuticareas wherein such inhibition is considered beneficial, including thetreatment of cardiovascular disorders and erectile dysfunction.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to compounds having the generalstructural formula (I):

wherein R⁰, independently, is selected from the group consisting of haloand C₁₋₆alkyl;

R¹ is selected from the group consisting of hydrogen, C₁₋₆alkyl,C₂₋₆alkenyl, C₂₋₆alkynyl, haloC₁₋₆-alkyl, C₃₋₈cycloalkyl,C₃₋₈cycloalkylC₁₋₃alkyl, aryl-C₁₋₃alkyl, and heteroarylC₁₋₃alkyl;

R² is selected from the group consisting of an optionally substitutedmonocyclic aromatic ring selected from the group consisting of benzene,thiophene, furan, and pyridine, and an optionally substituted bicyclicring

wherein the fused ring A is a 5- or 6-membered ring, saturated orpartially or fully unsaturated, and comprises carbon atoms andoptionally one or two heteroatoms selected from oxygen, sulfur, andnitrogen;

R³ is hydrogen or C₁₋₃alkyl,

or R¹ and R³ together form a 3- or 4-membered alkyl or alkenyl componentof a 5- or 6-membered heterocyclic ring;

rings B and C form an optionally substituted fused ring structureselected from the group consisting of

R^(a) is selected from the group consisting of hydrogen, C₁₋₆alkyl,C₃₋₈cycloalkyl, aryl, arylC₁₋₃-alkyl, C₁₋₃alkylenearyl; and

q is 0, 1, 2, 3, or 4;

pharmaceutically acceptable salts and hydrates thereof.

As used herein, the term “alkyl” includes straight chained and branchedhydrocarbon groups containing the indicated number of carbon atoms,typically methyl, ethyl, and straight chain and branched propyl andbutyl groups. The hydrocarbon group can contain up to 16 carbon atoms.The term “alkyl” includes “bridged alkyl,” i.e., a C₆-C₁₆bicyclic orpolycyclic hydrocarbon group, for example, norbornyl, adamantyl,bicyclo[2.2.2]octyl, bicyclo[2.2.1]heptyl, bicyclo[3.2.1]octyl, ordecahydronaphthyl.

The term “cycloalkyl” is defined as a cyclic C₃-C₈ hydrocarbon group,e.g., cyclopropyl, cyclobutyl, cyclohexyl, and cyclopentyl.

The terms “alkenyl” and “alkynyl” are defined similarly as alkyl, exceptthe group has a carbon-carbon double bond or a carbon-carbon triplebond, respectively.

The term “alkylene” refers to an alkyl group having a substituent. Forexample, the term “C₁₋₃alkylenearyl” refers to an alkyl group containingone to three carbon atoms, and substituted with an aryl group. The term“alkenylene” as used herein is similarly defined, and contains theindicated number of carbon atoms and a carbon-carbon double bond, andincludes straight chained and branched alkenylene groups, likeethyenylene.

The term “halo” or “halogen” is defined herein to include fluorine,bromine, chlorine, and iodine.

The term “haloalkyl” is defined herein as an alkyl group substitutedwith one or more halo substituents, either fluoro, chloro, bromo, iodo,or combinations thereof. Similarly, “halocycloalkyl” is defined as acycloalkyl group having one or more halo substituents.

The term “aryl,” alone or in combination, is defined herein as amonocyclic or polycyclic aromatic group, preferably a monocyclic orbicyclic aromatic group, e.g., phenyl or naphthyl, that can beunsubstituted or substituted, for example, with one or more, and inparticular one to three, alkyl, halo, hydroxy, hydroxyalkyl, alkoxy,alkoxyalkyl, haloalkyl, nitro, amino, alkylamino, acylamino, alkylthio,alkylsulfinyl, and alkylsulfonyl. Examples of aryl groups include, butare not limited to, phenyl, naphthyl, tetrahydronaphthyl,2-chlorophenyl, 3-chlorophenyl, 4-chlorophenyl, 2-methylphenyl,4-methoxyphenyl, 3-trifluoromethylphenyl, 4-nitrophenyl, and the like.

The term “heteroaryl” is defined herein as a monocyclic or bicyclic ringsystem containing one or two aromatic rings and containing at least onenitrogen, oxygen, or sulfur atom in an aromatic ring, and which can beunsubstituted or substituted, for example, with one or more, and inparticular one to three, substituents, like halo, alkyl, hydroxy,hydroxyalkyl, alkoxy, alkoxyalkyl, haloalkyl, nitro, amino, alkylamino,acylamino, alkylthio, alkylsulfinyl, and alkylsulfonyl. Examples ofheteroaryl groups include, but are not limited to, thienyl, furyl,pyridyl, oxazolyl, quinolyl, isoquinolyl, indolyl, triazolyl,isothiazolyl, isoxazolyl, imidizolyl, benzothiazolyl, pyrazinyl,pyrimidinyl, thiazolyl, and thiadiazolyl.

The term “hydroxy” is defined as —OH.

The term “hydroxyalkyl” is defined as a hydroxy group appended to analkyl group.

The term “alkoxy” is defined as —OR, wherein R is alkyl.

The term “alkoxyalkyl” is defined as an alkyl group wherein a hydrogenhas been replaced by an alkoxy group.

The term “amino” is defined as —NH₂, and the term “alkylamino” isdefined as —NR₂, wherein at least one R is alkyl and the second R isalkyl or hydrogen.

The term “acylamino” is defined as RC(═O)N, wherein R is alkyl or aryl.

The term “alkylthio” is defined as —SR, wherein R is alkyl.

The term “alkylsulfinyl” is defined as R—SO₂, where R is alkyl.

The term “alkylsulfonyl” is defined as R—SO₃, where R is alkyl.

The term “nitro” is defined as —NO₂.

The term “cyano” is defined as —CN.

With respect to R⁰, the R⁰ substituents can be present on the B ring orthe C ring. If more than one R⁰ is present, the R⁰ substituents can beon one ring or can be on both rings.

In a preferred embodiment, R² is the optionally substituted bicyclicring system

wherein the bicyclic ring can represent, for example, naphthalene orindene, or a heterocycle, such as benzoxazole, benzothiazole,benzisoxazole, benzimidazole, quinoline, indole, benzothiophene, orbenzofuran, or

wherein n is an integer 1 or 2, and X, independently, are C(R^(a))₂, O,S, or NR^(a). The bicyclic ring comprising the R² substituent typicallyis attached to the rest of the molecule by a phenyl ring carbon atom.

In a preferred group of compounds of formula (I), R² is represented byan optionally substituted bicyclic ring

wherein n is 1 or 2, and X, independently, are CH₂ or O. Especiallypreferred R² substituents include

Within this particular group of compounds, nonlimiting examples ofsubstituents for the bicyclic ring include halogen (e.g., chlorine),C₁₋₃alkyl (e.g., methyl, ethyl, or i-propyl), OR^(a) (e.g., methoxy,ethoxy, or hydroxy), CO₂R^(a), halomethyl or halomethoxy (e.g.,trifluoromethyl or trifluoromethoxy), cyano, nitro, and N(R^(a))₂.

An especially preferred subclass of compounds within the general scopeof formula (I) is represented by compounds of formula (II)

and pharmaceutically acceptable salts and solvates (e.g., hydrates)thereof.

Compounds of formula (I) can contain one or more asymmetric center, and,therefore, can exist as stereoisomers. The present invention includesboth mixtures and separate individual stereoisomers of the compounds offormula (I). Compounds of formula (I) also can exist in tautomericforms, and the invention includes both mixtures and separate individualtautomers thereof.

Pharmaceutically acceptable salts of the compounds of formula (I) can beacid addition salts formed with pharmaceutically acceptable acids.Examples of suitable salts include, but are not limited to, thehydrochloride, hydrobromide, sulfate, bisulfate, phosphate, hydrogenphosphate, acetate, benzoate, succinate, fumarate, maleate, lactate,citrate, tartrate, gluconate, methanesulfonate, benzenesulfonate, andp-toluenesulfonate salts. The compounds of the formula (I) also canprovide pharmaceutically acceptable metal salts, in particular alkalimetal salts and alkaline earth metal salts, with bases. Examples includethe sodium, potassium, magnesium, and calcium salts.

Compounds of the present invention are potent and selective inhibitorsof cGMP-specific PDE5. Thus, compounds of formula (I) are of interestfor use in therapy, specifically for the treatment of a variety ofconditions where selective inhibition of PDE5 is considered to bebeneficial.

Phosphodiesterases (PDEs) catalyze the hydrolysis of cyclic nucleotides,such as cyclic adenosine monophosphate (cAMP) and cyclic guanosinemonophosphate (cGMP). The PDEs have been classified into at least sevenisoenzyme families and are present in many tissues (J. A. Beavo,Physiol. Rev., 75, p. 725 (1995)).

PDE5 inhibition is a particularly attractive target. A potent andselective inhibitor of PDE5 provides vasodilating, relaxing, anddiuretic effects, all of which are beneficial in the treatment ofvarious disease states. Research in this area has led to several classesof inhibitors based on the cGMP basic structure (E. Sybertz et al.,Expert. Opin. Ther. Pat., 7, p. 631 (1997)).

The biochemical, physiological, and clinical effects of PDE5 inhibitorstherefore suggest their utility in a variety of disease states in whichmodulation of smooth muscle, renal, hemostatic, inflammatory, and/orendocrine function is desirable. The compounds of formula (I),therefore, have utility in the treatment of a number of disorders,including stable, unstable, and variant (Prinzmetal) angina,hypertension, pulmonary hypertension, chronic obstructive pulmonarydisease, congestive heart failure, malignant hypertension,pheochromocytoma, acute respiratory distress syndrome, acute and chronicrenal failure, atherosclerosis, conditions of reduced blood vesselpatency (e.g., postpercutaneous transluminal coronary or carotidangioplasty, or post-bypass surgery graft stenosis), peripheral vasculardisease, vascular disorders, such as Raynaud's disease, thrombocythemia,inflammatory diseases, myocardial infarction, stroke, bronchitis,chronic asthma, allergic asthma, allergic rhinitis, glaucoma,osteoporosis, preterm labor, benign prostatic hypertrophy, peptic ulcer,male erectile dysfunction, female sexual dysfunction, and diseasescharacterized by disorders of gut motility (e.g., irritable bowelsyndrome).

An especially important use is the treatment of male erectiledysfunction, which is one form of impotence and is a common medicalproblem. Impotence can be defined as a lack of power, in the male, tocopulate, and can involve an inability to achieve penile erection orejaculation, or both. The incidence of erectile dysfunction increaseswith age, with about 50% of men over the age of 40 suffering from somedegree of erectile dysfunction.

In addition, a further important use is the treatment of female arousaldisorder, also termed female sexual arousal disorder. Female sexualarousal disorders are defined as a recurrent inability to attain ormaintain an adequate lubrication/swelling response of sexual excitementuntil completion of sexual activity. The arousal response consists ofvasocongestion in the pelvis, vaginal lubrication, and expansion andswelling of external genitalia.

It is envisioned, therefore, that compounds of formula (I) are useful inthe treatment of male erectile dysfunction and female sexual arousaldisorder. Thus, the present invention concerns the use of compounds offormula (I), or a pharmaceutically acceptable salt thereof, or apharmaceutical composition containing either entity, for the manufactureof a medicament for the curative or prophylactic treatment of erectiledysfunction in a male animal and sexual arousal disorder in a femaleanimal, including humans.

The term “treatment” includes preventing, lowering, stopping, orreversing the progression or severity of the condition or symptoms beingtreated. As such, the term “treatment” includes both medical therapeuticand/or prophylactic administration, as appropriate.

It also is understood that “a compound of formula (I),” or aphysiologically acceptable salt or solvate thereof, can be administeredas the neat compound, or as a pharmaceutical composition containingeither entity.

Although the compounds of the invention are envisioned primarily for thetreatment of sexual dysfunction in humans, such as male erectiledysfunction and female sexual arousal disorder, they also can be usedfor the treatment of other disease states.

A further aspect of the present invention, therefore, is providing acompound of formula (I) for use in the treatment of stable, unstable,and variant (Prinzmetal) angina, hypertension, pulmonary hypertension,chronic obstructive pulmonary disease, congestive heart failure,malignant hypertension, pheochromocytoma, acute respiratory distresssyndrome, acute and chronic renal failure, atherosclerosis, conditionsof reduced blood vessel patency (e.g., post-PTCA or post-bypass graftstenosis), peripheral vascular disease, vascular disorders such asRaynaud's disease, thrombocythemia, inflammatory diseases, prophylaxisof myocardial infarction, prophylaxis of stroke, stroke, bronchitis,chronic asthma, allergic asthma, allergic rhinitis, glaucoma,osteoporosis, preterm labor, peptic ulcer, benign prostatic hypertrophy,male and female erectile dysfunction, or diseases-characterized bydisorders of gut motility (e.g., IBS).

According to another aspect of the present invention, there is providedthe use of a compound of formula (I) for the manufacture of a medicamentfor the treatment of the above-noted conditions and disorders.

In a further aspect, the present invention provides a method of treatingthe above-noted conditions and disorders in a human or nonhuman animalbody which comprises administering to said body a therapeuticallyeffective amount of a compound of formula (I).

Compounds of the invention can be administered by any suitable route,for example by oral, buccal, inhalation, sublingual, rectal, vaginal,transurethral, nasal, topical, percutaneous, i.e., transdermal, orparenteral (including intravenous, intramuscular, subcutaneous, andintracoronary) administration. Parenteral administration can beaccomplished using a needle and syringe, or using a high pressuretechnique, like POWDERJECT™.

Oral administration of a compound of the invention is the preferredroute. Oral administration is the most convenient and avoids thedisadvantages associated with other routes of administration. Forpatients suffering from a swallowing disorder or from impairment of drugabsorption after oral administration, the drug can be administeredparenterally, e.g., sublingually or buccally.

Compounds and pharmaceutical compositions suitable for use in thepresent invention include those wherein the active ingredient isadministered in an effective amount to achieve its intended purpose.More specifically, a “therapeutically effective amount” means an amounteffective to prevent development of, or to alleviate the existingsymptoms of, the subject being treated. Determination of the effectiveamounts is well within the capability of those skilled in the art,especially in light of the detailed disclosure provided herein.

A “therapeutically effective dose” refers to that amount of the compoundthat results in achieving the desired effect. Toxicity and therapeuticefficacy of such compounds can be determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, e.g., fordetermining the LD₅₀ (the dose lethal to 50% of the population) and theED₅₀ (the dose therapeutically effective in 50% of the population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex, which is expressed as the ratio between LD₅₀ and ED₅₀. Compoundswhich exhibit high therapeutic indices are preferred. The data obtainedfrom such data can be used in formulating a dosage range for use inhumans. The dosage of such compounds preferably lies within a range ofcirculating concentrations that include the ED₅₀ with little or notoxicity. The dosage can vary within this range depending upon thedosage form employed, and the route of administration utilized.

The exact formulation, route of administration, and dosage can be chosenby the individual physician in view of the patient's condition. Dosageamount and interval can be adjusted individually to provide plasmalevels of the active moiety which are sufficient to maintain thetherapeutic effects.

The amount of composition administered is dependent on the subject beingtreated, the subject's weight, the severity of the affliction, themanner of administration, and the judgment of the prescribing physician.

Specifically, for administration to a human in the curative orprophylactic treatment of the conditions and disorders identified above,oral dosages of a compound of formula (I) generally are about 0.5 toabout 1000 mg daily for an average adult patient (70 kg). Thus, for atypical adult patient, individual tablets or capsules contain 0.2 to 500mg of active compound, in a suitable pharmaceutically acceptable vehicleor carrier, for administration in single or multiple doses, once orseveral times per day. Dosages for intravenous, buccal, or sublingualadministration typically are 0.1 to 500 mg per single dose as required.In practice, the physician determines the actual dosing regimen which ismost suitable for an individual patient, and the dosage varies with theage, weight, and response of the particular patient. The above dosagesare exemplary of the average case, but there can be individual instancesin which higher or lower dosages are merited, and such are within thescope of this invention.

For human use, a compound of the formula (I) can be administered alone,but generally is administered in admixture with a pharmaceutical carrierselected with regard to the intended route of administration andstandard pharmaceutical practice. Pharmaceutical compositions for use inaccordance with the present invention thus can be formulated in aconventional manner using one or more physiologically acceptablecarriers comprising excipients and auxiliaries that facilitateprocessing of compounds of formula (I) into preparations which can beused pharmaceutically.

These pharmaceutical compositions can be manufactured in a conventionalmanner, e.g., by conventional mixing, dissolving, granulating,dragee-making, levigating, emulsifying, encapsulating, entrapping, orlyophilizing processes. Proper formulation is dependent upon the routeof administration chosen. When a therapeutically effective amount of acompound of the present invention is administered orally, thecomposition typically is in the form of a tablet, capsule, powder,solution, or elixir. When administered in tablet form, the compositioncan additionally contain a solid carrier, such as a gelatin or anadjuvant. The tablet, capsule, and powder contain about 5% to about 95%compound of the present invention, and preferably from about 25% toabout 90% compound of the present invention. When administered in liquidform, a liquid carrier such as water, petroleum, or oils of animal orplant origin can be added. The liquid form of the composition canfurther contain physiological saline solution, dextrose or othersaccharide solutions, or glycols. When administered in liquid form, thecomposition contains about 0.5% to about 90% by weight of a compound ofthe present invention, and preferably about 1% to about 50% of acompound of the present invention.

When a therapeutically effective amount of a compound of the presentinvention is administered by intravenous, cutaneous, or subcutaneousinjection, the composition is in the form of a pyrogen-free,parenterally acceptable aqueous solution. The preparation of suchparenterally acceptable solutions, having due regard to pH, isotonicity,stability, and the like, is within the skill in the art. A preferredcomposition for intravenous, cutaneous, or subcutaneous injectiontypically contains, in addition to a compound of the present invention,an isotonic vehicle.

For oral administration, the compounds can be formulated readily bycombining a compound of formula (I) with pharmaceutically acceptablecarriers well known in the art. Such carriers enable the presentcompounds to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions and the like, for oralingestion by a patient to be treated. Pharmaceutical preparations fororal use can be obtained by adding a compound of formula (I) with asolid excipient, optionally grinding a resulting mixture, and processingthe mixture of granules, after adding suitable auxiliaries, if desired,to obtain tablets or dragee cores. Suitable excipients include, forexample, fillers and cellulose preparations. If desired, disintegratingagents can be added.

For administration by inhalation, compounds of the present invention areconveniently delivered in the form of an aerosol spray presentation frompressurized packs or a nebulizer, with the use of a suitable propellant.In the case of a pressurized aerosol, the dosage unit can be determinedby providing a valve to deliver a metered amount. Capsules andcartridges of, e.g., gelatin, for use in an inhaler or insufflator canbe formulated containing a powder mix of the compound and a suitablepowder base such as lactose or starch.

The compounds can be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection can be presented in unit dosage form, e.g., in ampules orin multidose containers, with an added preservative. The compositionscan take such forms as suspensions, solutions, or emulsions in oily oraqueous vehicles, and can contain formulatory agents such as suspending,stabilizing, and/or dispersing agents.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds can be prepared asappropriate oily injection suspensions. Suitable lipophilic-solvents orvehicles include fatty oils or synthetic fatty acid esters. Aqueousinjection suspensions can contain substances which increase theviscosity of the suspension. Optionally, the suspension also can containsuitable stabilizers or agents that increase the solubility of thecompounds and allow for the preparation of highly concentratedsolutions. Alternatively, a present composition can be in powder formfor constitution with a suitable vehicle, e.g., sterile pyrogen-freewater, before use.

Compounds of the present invention also can be formulated in rectalcompositions, such as suppositories or retention enemas, e.g.,containing conventional suppository bases. In addition to theformulations described previously, the compounds also can be formulatedas a depot preparation. Such long-acting formulations can beadministered by implantation (for example, subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds can be formulated with suitable polymeric or hydrophobicmaterials (for example, as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

Many of the compounds of the present invention can be provided as saltswith pharmaceutically compatible counterions. Such pharmaceuticallyacceptable base addition salts retain the biological effectiveness andproperties of the free acids, and are obtained by reaction with suitableinorganic or organic bases.

In particular, a compound of formula (I) can be administered orally,buccally, or sublingually in the form of tablets containing excipients,such as starch or lactose, or in capsules or ovules, either alone or inadmixture with excipients, or in the form of elixirs or suspensionscontaining flavoring or coloring agents. Such liquid preparations can beprepared with pharmaceutically acceptable additives, such as suspendingagents. A compound also can be injected parenterally, for example,intravenously, intramuscularly, subcutaneously, or intracoronarily. Forparenteral administration, the compound is best used in the form of asterile aqueous solution which can contain other substances, forexample, salts or monosaccharides, such as mannitol or glucose, to makethe solution isotonic with blood.

For veterinary use, a compound of formula (I) or a nontoxic saltthereof, is administered as a suitably acceptable formulation inaccordance with normal veterinary practice. The veterinarian can readilydetermine the dosing regimen and route of administration that is mostappropriate for a particular animal.

Thus, the invention provides in a further aspect a pharmaceuticalcomposition comprising a compound of the formula (I), together with apharmaceutically acceptable diluent or carrier therefor. There isfurther provided by the present invention a process of preparing apharmaceutical composition, comprising a compound of formula (I), whichprocess comprises mixing a compound of formula (I), together with apharmaceutically acceptable diluent or carrier therefor.

In a particular embodiment, the invention includes a pharmaceuticalcomposition for the curative or prophylactic treatment of erectiledysfunction in a male animal, or sexual arousal disorder in a femaleanimal, including humans, comprising a compound of formula (I) or apharmaceutically acceptable salt thereof, together with apharmaceutically acceptable diluent or carrier.

Compounds of formula (I) can be prepared by any suitable method known inthe art, or by the following processes which form part of the presentinvention. In the synthetic methods below, R⁰, R¹, R², and R³ are asdefined in structural formula (I) above. For example, compounds offormula (I) can be prepared by the methods set forth in Daugan U.S. Pat.No. 5,859,006, incorporated herein by reference, utilizing theappropriate starting materials. Protecting compounds and protectinggroups, like benzyl chloroformate and trichloroethyl chloroformate, thatare well known to persons skilled in the art, for example, see T. W.Greene et al. “Protective Groups in Organic Synthesis, Third Edition,”John Wiley and Sons, Inc., NY, N.Y. (1999), also can be utilized in thesynthesis of compounds of structural formula (I).

Compounds of formula (I) can be converted to other compounds of formula(I). Thus, for example, when R² is a substituted benzene ring, it ispossible to prepare another suitably substituted compound of formula(I). Examples of appropriate interconversions include, but are notlimited to, nitro to amino, OR^(a) to hydroxy by suitable reducing means(e.g., using a suitable agent, such as SnCl₂ or a palladium catalyst,such as palladium-on-carbon), or amino to substituted amino, such asacylamino or sulphonylamino, using standard acylating or sulfonylatingconditions. In cases wherein R² represents a substituted bicyclicsystem, suitable interconversion can involve removal of a substituent,such as by treatment with a palladium catalyst whereby, for example, abenzyl substituent is removed from a suitable bicyclic system.

Compounds of formula (I) can be prepared by the method above asindividual stereoisomers from the appropriate stereoisomer or as aracemic mixture. Individual stereoisomers of the compounds of theinvention can be prepared from racemates by resolution using methodsknown in the art for the separation of racemic mixtures into theirconstituent stereoisomers, for example, using HPLC on a chiral column,such as Hypersil naphthyl urea, or using separation of salts ofstereoisomers. Compounds of the invention can be isolated in associationwith solvent molecules by crystallization from, or evaporation of, anappropriate solvent.

The pharmaceutically acceptable acid addition salts of the compounds offormula (I) that contain a basic center can be prepared in aconventional manner. For example, a solution of the free base can betreated with a suitable acid, either neat or in a suitable solution, andthe resulting salt isolated either by filtration or by evaporation undervacuum of the reaction solvent. Pharmaceutically acceptable baseaddition salts can be obtained in an analogous manner by treating asolution of a compound of formula (I) with a suitable base. Both typesof salt can be formed or interconverted using ion-exchange resintechniques. Thus, according to a further aspect of the invention, amethod for preparing a compound of formula (I) or a salt or solvate(e.g., hydrate) is provided, followed by (i) salt formation, or (ii)solvate (e.g., hydrate) formation.

The following abbreviations are used hereafter in the accompanyingexamples: rt (room temperature), min (minute), h (hour), g (gram), mmol(millimole), m.p. (melting point), eq (equivalents), L (liter), mL(milliliter), μL (microliters), Me (methyl), Bn (benzyl), DMSO (dimethylsulfoxide), CH₂Cl₂ (dichloromethane), IPA (isopropyl alcohol), TFA(trifluoroacetic acid), TPAP (tetrapropylammonium perruthenate), SOCl₂(thionyl chloride), Et₃N (triethylamine), CH₃NH₂ (methylamine), EtOAc(ethyl acetate), EtOH (ethanol), DMF (dimethyl formamide), CHCl₃(chloroform), EtOAc (ethyl acetate), MeOH (methanol), and THF(tetrahydrofuran).

PREPARATION OF EXAMPLE 1

Example 1

Example 1 was prepared from Intermediates 1-3 as depicted in thefollowing synthetic scheme. Intermediate 1 was prepared fromDL-7-azatryptophan, a commercially available compound from AldrichChemical Co., Milwaukee, Wis.

Intermediate 1 Preparation of DL-7-Azatryptophan Methyl EsterMonohydrochloride

Thionyl chloride (1.74 g, 1.1 mL, 14.6 mmol) was added dropwise to asuspension of DL-7-azatryptophan monohydrate (1.00 g, 4.87 mmol) inanhydrous methanol (40 mL) at 0° C. under a nitrogen blanket. Themixture was warmed slowly to room temperature and stirred for 24 hours.The methanol was removed under reduced pressure to provide a whitesolid. Analysis of the resulting solid by ¹H NMR showed the solid to bea mixture of starting material and Intermediate 1. The thionyl chloridetreatment was repeated an additional time as described above to providea white solid (1.39 g, 100%). ¹H NMR (300 MHz, CDCl₃): δ 12.05 (s, 1H),8.54 (bs, 2H), 8.31 (d, J=4.3 Hz, 1H), 8.18 (d, J=8.0 Hz, 1H), 7.47 (d,J=2.1 Hz, 1H), 7.24 (dd, J=5.0 Hz, J=7.8 Hz, 1H), 4.34 (m, 1H), 3.70 (s,3H), 3.33 (d, J=6.1 Hz, 2H).

Intermediate 2 Preparation of a (+/−)-cis-β-Carboline

A suspension of Intermediate 1 (1.39 g, 5.44 mmol) and piperonal (1.06g, 7.07 mmol) in pyridine (35 mL) was warmed to 100° C. and stirred for5 hours under a nitrogen blanket. The resulting brown suspension wascooled to room temperature and filtered to remove unreacted startingmaterial. The filtrate was concentrated in vacuo and was purified bycolumn chromatography (silica gel, 1-20% MeOH/CH₂Cl₂) to give 0.34 g ofa white solid. The solid was repurified by column chromatography (silicagel, 0-4% MeOH/CHCl₃), and was triturated with MeOH to provide 0.31 g(16.4%) of the cis product, which was approximately 94% pure by ¹H NMRanalysis. TLC R_(f) (5% MeOH/CH₂Cl₂)=0.51; ¹H NMR (306 MHz, CDCl₃): δ10.99 (s, 1H), 8.10 (dd, J=1.4 Hz, J=4.7 Hz, 1H), 7.86 (d, J=6.75, 1H),7.00 (dd, J=4.7 Hz, J=7.8 Hz, 1H), 6.81-6.90 (m, 3H), 6.00 (d, J=1 Hz,1H), 5.16 (m, 1H), 3.82-3.89 (m, 1H), 3.70 (s, 3H), 2.98-3.07 (m, 1H),2.77-2.87 (m, 1H), 2.68-2.73 (m, 1H); MS (API) m/z 352 (M+H). The transcarboline also was eluted from the column in impure form: TLC R_(f) (5%MeOH/CH₂Cl₂)=0.40.

Intermediate 3 Preparation of a (+/−)-cis-2-Chloroacetyl-β-carboline

Chloroacetyl chloride (0.11 mL, 1.34 mmol) was added dropwise to amixture of Intermediate 2 (0.31 g, 0.89 mmol) and triethylamine (0.5 mL,3.6 mmol) in CH₂Cl₂ (25 mL) at 0° C. under a nitrogen blanket. Theresulting mixture was warmed to room temperature and stirred for about1.5 hours. The reaction was quenched with 1 N HCl (2 mL), and wasdiluted with CH₂Cl₂ (25 mL) and water (10 mL). The layers wereseparated. The organic phase was washed with saturated sodiumbicarbonate (NaHCO₃) and brine, then dried over anhydrous sodium sulfate(Na₂SO₄). Filtration and concentration in vacuo yielded Intermediate 3as a greenish foam (0.39 g), which was used without purification in thenext step: TLC R_(f) (10% MeOH/CH₂Cl₂)=0.58; MS (API) m/z 428 (M+H).

Example 1 Preparation of(+/−,cis)-10-Benzo[1,3]dioxol-5-yl-7-methyl-5,5a,7,8,10,11-hexahydro-1,7,9a,11-tetraazabeno[β]fluorene-6,9-dione

A mixture of the crude Intermediate 3 (0.38 g, 0.89 mmol), 40%methylamine in water (1.47 mL, 17.1 mmol), THF (40 mL), and MeOH (10 mL)was heated at 40° C. under a nitrogen blanket for 2.5 hours. Theresulting solution was cooled to room temperature, then acidified withconcentrated hydrochloric acid (HCl). The resulting mixture was dilutedwith CH₂Cl₂ (25 mL), the layers were separated, and the organic phasewas washed with water (10 mL) and brine (10 mL), The organic layer wasdried over anhydrous Na₂SO₄, filtered and concentrated in vacuo. Theproduct was purified by column chromatography (silica gel, 0-5%MeOH/CH₂Cl₂) to provide a white solid (0.20 g, 57.5% over two steps)after drying at 45° C. under vacuum. The product was contaminated withapproximately 10% of the trans isomer: mp 262-276° C.; TLC R_(f) (10%MeOH/CH₂Cl₂)=0.45; ¹H NMR (300 MHz, DMSO-d₆): δ 11.65 (s, 1H), 8.15 (dd,J=1.5 Hz, J=4.7 Hz, 1H), 8.00 (dd, J=1.2 Hz, J=7.8 Hz, 1H), 7.05 (dd,J=4.8 Hz, J=7.9 Hz, 1H), 6.90 (s, 1H), 6.82-6.90 (m, 2H), 6.13 (s, 1H),5.93 (s, 2H), 4.42 (dd, J=4.4 Hz, J=11.8 Hz, 1H), 4.19 (d, J=16.6 Hz,1H), 3.96 (d, J=17.2 Hz, 1H), 3.55 (dd, J=4.51, J=16.0 Hz, 1H),2.92-3.01 (m, 4H), 3.94 (s, 3H); MS (API) m/z 391 (M+H); [α]_(D)^(25° C.)=no observed rotation (c=0.15, DMSO). Anal. Calcd. forC₂₁H₁₈N₄O₄.0.7 H₂O: C, 62.59; H, 4.85; N, 13.90. Found: C, 62.61; H,4.71; N, 13.88. The relative stereochemistry of the major product wasconfirmed to be the cis isomer by NOE difference experiments (DMSO-d₆):positive NOE enhancements from the C5a proton at 4.42 ppm to the C10proton at 6.13 ppm.

PREPARATION OF EXAMPLE 2

Example 2

Example 2 can be prepared from the indolizine-substituted alanineIntermediate 4, the synthesis of which is disclosed in M. Cardellini etal., Ann. Chim. (Rome), 58(11), pages 1206-1213 (1968), and P. Gmeineret al., Arch. Pharm., 321(9), pages 505-507 (1988). The following is anenvisioned synthetic route to Example 2.

PREPARATION OF EXAMPLE 3

The following Example 3 can be prepared by the same envisioned syntheticroute as Example 2, starting with the azaindolizine-substituted alanineIntermediate 5.

Example 3 PREPARATION OF EXAMPLE 4

Example 4

Example 4 was prepared from Intermediates 6-8 as depicted in thefollowing synthetic scheme. Intermediate 6 was prepared from(R)-α-amino-3-[1]benzothiophene-3-ylpropionic acid, commerciallyavailable from Aldrich Chemical Co., Milwaukee, Wis. Aspects of thefollowing sequence are disclosed in H. Kawakubo, J. Med. Chem., 36,pages 3526-3532 (1993) and H. Kawakubo et al., J. Med. Chem., 33, pages3110-3116 (1990).

Intermediate 6 Preparation of(R)-α-Amino-3-[1]benzothiophene-3-ylpropionic Acid Methyl Ester

Thionyl chloride (2.69 g, 22.6 mmol) was added dropwise to a suspensionof (R)-α-amino-3-[1]benzothiophene-3-ylpropionic acid (1.00 g, 4.52mmol) in anhydrous MeOH (20 mL) at 0° C. under a nitrogen blanket. Themixture was warmed slowly to room temperature and stirred for a total of24 hours. The solvent was removed under reduced pressure to provide apale yellow solid. The resulting residue was dissolved in MeOH (10 mL),then diluted with 1:1 CH₂Cl₂:saturated NaHCO₃ (50 mL). The layers wereseparated, and the aqueous layer was back-extracted with CH₂Cl₂ (25 mL).The combined organic phases were washed with water, saturated sodiumchloride (NaCl), dried over anhydrous Na₂SO₄, filtered, and concentratedto provide a pale brown oil (1.08 g, 100%): TLC R_(f) (90:10:1;CH₂Cl₂:EtOAc:MeOH)=0.34; ¹H NMR (300 MHz, CDCl₃): δ 7.88-7.78 (m, 2H),7.43-7.34 (m, 2H), 7.26 (s, 1H), 3.88 (dd, J=5.0 Hz, J=8.1 Hz, 1H), 3.72(s, 3H), 3.41 (dd, J=0.7 Hz, J=4.9 Hz, 1H), 3.36 (dd, J=0.7 Hz, J=4.9Hz, 1H), 3.10 (dd, J=8.4 Hz, J=14.1 Hz, 1H).

Intermediate 7 Preparation of a (+/−)-cis-β-carboline

Trifluoroacetic acid (2.4 mL, 33.2 mmol) was added to a mixture ofIntermediate 6 (2.37 g, 5.24 mmol) and piperonal (1.08 g, 7.21 mmol) intoluene (20 mL). The mixture was warmed to 40° C. and stirred for atleast 24 hours. The solution was cooled to room temperature, dilutedwith ethyl acetate (200 mL) and washed with 1N HCl (20 mL), saturatedNaHCO₃ (2×30 mL), and saturated NaCl (30 mL). The organic phase wasdried over anhydrous Na₂SO₄, filtered, and the solvent was removed underreduced pressure to provide a brown oily solid. The crude product waspurified by column chromatography (silica gel, 0-5% EtOAc/CH₂Cl₂) toprovide 0.33 g (16.9%) of a pale yellow foam. TLC R_(f) (5%EtOAc/CH₂Cl₂)=0.44; ¹H NMR (300 MHz, CDCl₃): δ 7.82 (d, J=7.7 Hz, 1H),7.71 (d, J=7.5 Hz, 1H), 7.41-7.28 (m, 2H), 6.96-6.88 (m, 3H), 6.01 (d,J=0.8 Hz, 2H), 5.19 (m, 1H), 3.99-3.96 (m, 1H), 3.74 (s, 3H), 3.17-3.11(m, 1H), 2.96 (m, 1H), 2.91-2.84 (m, 1H). The trans carboline was alsoeluted from the column in impure form: TLC R_(f) (5% EtOAc/CH₂Cl₂)=0.38.

Intermediate 8 Preparation of a (+/−)-cis-2-Chloroacetyl-β-carboline 4

Chloroacetyl chloride (0.09 mL, 1.16 mmol) was added dropwise to amixture of Intermediate 7 (0.33 g, 0.89 mmol) and triethylamine (0.25mL, 1.78 mmol) in anhydrous THF (8 mL) at 0° C. under a nitrogenblanket. The resulting mixture was warmed to room temperature andstirred for 0.5 hour. The reaction was quenched with 1N HCl (3 mL) andwas concentrated in vacuo. The residue was taken up in CH₂Cl₂ (50 mL),then washed with water (2×10 mL) and saturated NaCl (10 mL), and driedover anhydrous Na₂SO₄. Filtration and concentration in vacuo provided0.44 g of a beige foam, which was used without purification in the nextstep: TLC R_(f) (3% EtOAc/CH₂Cl₂)=0.57.

Example 4 Preparation of(+/−,cis)-10-Benzo[1,3]dioxol-5-yl-7-methyl-5,7,8,10-tetrahydro-5aH-11-thia-7,9a-diazabenzo[β]fluorene-6,9-dione

A mixture of crude Intermediate 8 (about 0.39 g, 0.89 mmol), 40%methylamine in water (0.38 mL, 4.45 mmol) in THF (2 mL) was heated at45° C. under a nitrogen blanket for 15 minutes. The resulting solutionwas cooled to room temperature, quenched with 0.2 mL concentrated HCl.The THF was removed by vacuum distillation to yield a beige solid. Thesolid was collected by filtration and reslurried in MeOH. Filtration andwashing with MeOH (2×10 mL) provided a cream solid (0.23 g, 63% for twosteps); mp 245-266° C.; TLC R_(f) (5% EtOAc/CH₂Cl₂)=0.12; ¹H NMR (300MHz, DMSO-d₆): δ 7.88 (dd, J=7.7 Hz, J=14.9 Hz, 2H), 7.42 (dt, J=1.0 Hz,J=7.6 Hz, 1H), 7.35 (dt, J=1.2 Hz, J=7.5 Hz, 1H), 6.87 (s, 1H),6.76-6.82 (m, 2H), 6.25 (s, 1H), 5.93 (d, J=1.4 Hz, 2H), 4.48 (dd, J=4.1Hz, J=11.7 Hz, 1H), 4.19 (d, J=16.6 Hz, 1H), 3.96 (d, J=17.2 Hz, 1H),3.68 (dd, J=4.4 Hz, J=16.3 Hz, 1H), 3.08-2.99 (m, 1H), 2.94 (s, 3H); MS(API) m/z 407 (M+H), 429 (M+Na); [α]_(D) ^(25° C.)=no observed rotation(c=0.51, DMSO). Anal. Calcd. for C₂₂H₁₈N₂O₄S₁.0.5H₂O; C, 63.60; H, 4.61;N, 6.74; S, 7.72. Found: C, 63.73; H, 4.62; N, 6.71; S, 7.93. Therelative stereochemistry of the product was confirmed to be the cisisomer by NOE difference experiments (DMSO-d₆): positive NOEenhancements from the C12a proton at 4.48 ppm to the C6 proton at 6.25ppm.

PREPARATION OF EXAMPLES 5(a) AND 5(b) AND EXAMPLES 6 AND 7

Example 5(a)

Example 5(b)

Example 6 (X═O) Example 7 (X═S)

Examples 5(a), 5(b), 6, and 7 were prepared from the followingcommercially available compounds:

wherein X is NH, O, or S. The synthesis of 2-indolyl alanine,2-benzofuranyl alanine, and 2-benzothiophenyl alanine starting materialsalso are disclosed in T. Masquelin et al., Helv. Chim. Acta, 77, pages1395-1411 (1994). The following scheme illustrates one envisionedpreparation of Example 5. The compounds of Examples 6 and 7 can besynthesized analogously.

Example 5

The following illustrates another synthetic sequence to Examples 5(a)and 5(b).

PREPARATION OF EXAMPLE 5(a)

Example 5(a)

Example 5(a) was prepared from the following Intermediates 9 and 11 asdepicted in the following synthetic scheme. Intermediates 9 and 10 wereprepared from D-isotryptophan, a commercially available compound.

Intermediates 9 and 10 Preparation of cis-β-Carboline andtrans-β-Carboline

To a solution of D-isotryptophan (0.84 g, 3.6 mmol) and piperonal (0.71g, 4.8 mmol) in methylene chloride (30 mL) at 0° C. under an argonblanket was added trifluoroacetic acid (0.56 mL, 7.2 mmol). Then themixture was warmed to room temperature over 4 hours. The resulting pinksolution was adjusted to a basic pH with saturated sodium bicarbonatesolution. The phases were separated, and the aqueous phase was extractedwith methylene chloride (2×50 mL). The combined organic phases weredried over Na₂SO₄, then the solvent was removed under reduced pressureto provide a pale pink oil as a residue. The residue was purified byflash column chromatography, eluting with methylene chloride/ethylacetate (6:1), to provide cis-β-carboline Intermediate 9 as a yellow oil(0.55 g, 42%): TLC R_(f) (6:1 methylene chloride/ethyl acetate)=0.62; ¹HNMR (300 MHz, CDCl₃): δ 7.90 (s, H), 7.30 (d, J=8.1 Hz, 1H), 7.07 (t,J=7.5 Hz, 1H), 6.94-6.75 (m, 5H), 5.92 (d, J=1.4 Hz, 2H), 5.18 (s, 1H),4.30-4.19 (m, 2H), 3.97-3.92 (dd, J=6.3, 8.8 Hz, 1H), 3.13-3.09 (m, 2H),1.31 (t, J=7.1 Hz, 3H) ppm. The latter eluting trans isomer Intermediate10 also was isolated as a yellow oil (0.73 g, 55%): TLC R_(f) (6:1methylene chloride/ethyl acetate)=0.38; ¹H NMR (300 MHz, CDCl₃): δ 8.04(s, 1H), 7.29 (d, J=8.1 Hz, 1H), 7.12-7.07 (m, 1H), 7.02-6.91 (m, 2H),6.82-6.72 (m, 3H), 5.91 (d, J=1.6 Hz, 2H), 5.35 (s, 1H), 4.27-4.07 (m,2H), 3.96-3.92 (dd, J=5.3, 7.2 Hz, 1H), 3.17-3.07 (m, 2H), 2.35 (bs,1H), 1.27 (t, J=7.1 Hz, 3H) ppm.

Intermediate 11 Preparation of cis-2-Chloroacetyl-β-carboline

Chloroacetyl chloride (0.16 mL, 2.01 mmol) was added to a solution ofcis-β-carboline Intermediate 9 (0.55 g, 1.51 mmol) and triethylamine(0.30 mL, 2.15 mmol) in methylene chloride (40 mL) at 0° C. under anargon blanket, after which the resulting mixture was warmed to roomtemperature over 3 hours. The yellow slurry was dissolved in methylenechloride (50 mL), then washed with water (20 mL) and saturated NaHCO₃solution (20 mL). The organic phase was dried over Na₂SO₄, then thesolvent was removed under reduced pressure to yieldcis-2-chloroacetyl-β-carboline Intermediate 11 as an orange solid whichwas used without further purification (0.62 g, 93%): TLC R_(f) (8:1methylene chloride/ethyl acetate)=0.63.

Example 5(a) Preparation of(5R,9aR)-5-Benzo(1,3)dioxol-5-yl-8-methyl-5,7,8,9a,10,11-hexahydro-5a,8,11-triaza-benzo[b]fluorane-8,9-dione

A solution of Intermediate 11 (0.62 g, 1.41 mmol) and methylamine (3.5mL, 7.03 mmol, 2 M solution in THF) in methanol (15 mL) was heated at50° C. under an argon blanket for 16 hours. The resulting solids wereisolated by filtration under reduced pressure to provide Example 5(a) asa pale yellow solid (0.207 g, 38%): mp 274-277° C.; TLC R_(f) (4:1:0.3methylene chloride/ethyl acetate/methanol)=0.40; ¹H NMR (300 MHz,DMSO-d₆): δ 11.14 (s, H), 7.45 (d, J=7.8 Hz, 1H), 7.30 (d, J=7.9 Hz,1H), 7.05-6.99 (dt, J=1.0, 7.5 Hz, 1H), 6.92 (t, J=7.5 Hz, 1H), 6.87 (s,1H), 6.83 (d, J=1.7 Hz, 1H), 6.71 (d, J=7.7 Hz, 1H), 6.23 (s, 1H),5.88-5.87 (dd, J=1.0, 3.7 Hz, 2H), 4.51-4.45 (dd, J=4.3, 11.5 Hz, 1H),4.16 (d, J=17.3 Hz, 1H), 3.92 (d, J=17.2 Hz, 1H), 3.47-3.40 (dd, J=4.7,16.4 Hz, 1H), 3.32-3.22 (m, 1H), 2.92 (s, 3H) ppm; ESI MS m/z 390[C₂₂H₁₉N₃O₄+H]⁺. Anal. Calcd. for C₂₂H₁₉N₃O₄: C, 67.86; H, 4.92; N,10.79. Found: C, 67.53; H, 4.96; N, 10.73. HPLC analysis (Chiralcel ODColumn, 250×4.5 mm, Retention Time=12.8 min; 1:1 isopropanol/-hexane;flow=1.00 mL/min; detector 254 nm; temperature ambient) showed one peak,with a purity of 99.5%. The stereochemistry of Example 5(a) wasconfirmed to be the desired cis isomer by a series of NOE differenceexperiments: a positive NOE enhancement from the C12a proton at 4.50 ppmto the C6 proton at 6.23 ppm; a positive NOE enhancement from the C6proton at 6.23 ppm to the C12a proton at 4.50 ppm.

PREPARATION OF EXAMPLE 5(b)

Example 5(b)

Example 5(b) was prepared from the above-described Intermediate 10 asdepicted in the following synthetic scheme.

Intermediate 12 Preparation of (+)-trans-2-Chloroacetyl-β-carboline

Chloroacetyl chloride (0.21 mL, 2.64 mmol) was added to a solution ofIntermediate 10 (0.73 g, 2.00 mmol) and triethylamine (0.36 mL, 2.58mmol) in methylene chloride (45 mL) at 0° C. under an argon blanket,after which the resulting mixture was warmed to room temperature over 3hours. The orange solution was dissolved in methylene chloride (50 mL),washed with water (20 mL) and a saturated NaHCO₃ solution (20 mL). Theorganic phase was dried over Na₂SO₄, then the solvent was removed underreduced pressure to afford trans-2-chloroacetyl-β-carboline Intermediate12 as an orange foam, which was used without further purification (0.86g, 98%): TLC R_(f) (8:1 methylene chloride/ethyl acetate)=0.48.

Example 5(b) Preparation of(5S,9aR)-5-Benzo[1,3]dioxol-5-yl-8-methyl-5,7,8,9a,10,11-hexahydro-5a,8,11-triaza-benzo[b]fluorene-8,9-dione

A solution of trans-2-chloroacetyl-β-carboline Intermediate 12 (0.86 g,1.95 mmol) and methylamine (9.8 mL, 19.5 mmol, 2 M solution in THF) inmethanol (15 mL) was heated at 50° C. under an argon blanket for 16hours. The resulting solution was cooled to room temperature, thenconcentrated under reduced pressure to provide an orange foam. Theresidue was purified by flash column chromatography, eluting withmethylene chloride/ethyl acetate/methanol (4:1:0.3) to provide a yellowfoam. The foam was slurried in diethyl ether and the solids werecollected by vacuum filtration to provide Example 5(b) as an off-whitesolid (0.345 g, 45%): mp 274-277° C.; TLC R_(f) (4:1:0.3 methylenechloride/ethyl acetate/methanol)=0.30; ¹H NMR (300 MHz, DMSO-d₆): δ11.19 (s, 1H), 7.36 (d, J=8.0 Hz, 1H), 7.08-7.00 (m, 2H), 6.91-6.80 (m,4H), 6.68 (d, J=8.1 Hz, 1H), 5.97 (d, J=4;2 Hz, 1H), 4.23 (d, J=17.5 Hz,1H), 4.15-4.09 (dd, J=4.6, 11.2 Hz, 1H), 4.03 (d, J=17.5 Hz, 1H),3.36-3.10 (m, 3H), 2.84 (s, 3H) ppm; ESI MS m/z 390 [C₂₂H₁₉N₃O₄+H]⁺.Anal. Calcd. for C₂₂H₁₉N₃O₄: C, 67.86; H, 4.92; N, 10.79. Found: C,67.49; H, 4.95; N, 10.68. HPLC analysis (Chiralcel OD Column 250×4.5 mm,Retention Times=8.5 and 10.9 min; 1:1 isopropanol/hexane; flow=1.00mL/min; detector 254 nm; temperature ambient) showed two peaks, with aratio of 5:95, respectively, and with a total purity of 100.0%. Thestereochemistry of Example 5(b) was confirmed to be the desired transisomer by a series of NOE difference experiments: no NOE enhancementfrom the C12a proton at 4.40 ppm to the C6 proton at 7.08 ppm; no NOEenhancement from the C6 proton at 7.08 ppm to the C12a proton at 4.40ppm.

PREPARATION OF EXAMPLE 8

Example 8

The envisioned synthesis of the Example 8 benzimidizole utilizes thesame sequence as in the synthesis of Examples 5-7 beginning withesterification of known Intermediate 13 amino acid in methanol catalyzedby sulfuric acid.

PREPARATION OF EXAMPLE 9

Example 9

Example 9 can be prepared from Intermediate 14, a 1-indolyl alaninedescribed in D. Ranganathan et al., Tetrahedron Lett., 23(27), pages2789-2792 (1982). The same synthetic sequence used to manufactureExample 8 can be utilized in the synthesis of Example 9.

PREPARATION OF EXAMPLE 10

Example 10

Synthesis of indolizine Example 10 from amino ester Intermediate 15utilizes the same synthetic scheme as Example 8. Intermediate 15 isprepared from known aldehyde Intermediate 16 by use of an appropriateWittig reaction followed by catalytic hydrogenation of the resultingalkene.

PREPARATION OF EXAMPLES 11 AND 12

Example 11

Example 12

Examples 11 and 12 were prepared from the following Intermediates 17 and18 by the same procedure utilized to prepare Example 8.

The following compound, Example 13, can be prepared by a sequencesimilar to the preparation of Examples 1-12:

Example 13

Compounds of the present invention can be formulated into tablets fororal administration. For example, a compound of formula (I) can beformed into a dispersion with a polymeric carrier by the coprecipitationmethod set forth in WO 96/38131, incorporated herein by reference. Thecoprecipitated dispersion then can be blended with excipients, thenpressed into tablets, which optionally are film-coated.

The compounds of structural formula (I) were tested for an ability toinhibit PDE5. The ability of a compound to inhibit PDE5 activity isrelated to the IC₅₀ value for the compound, i.e., the concentration ofinhibitor required for 50% inhibition of enzyme activity. The IC₅₀ valuefor compounds of structural formula (I) were determined usingrecombinant human PDE5.

The compounds of the present invention typically exhibit an IC₅₀ valueagainst recombinant human PDE5 of less than about 50 μM, and preferablyless than about 25 μM, and more preferably less than about 15 μm. Thecompounds of the present invention typically exhibit an IC₅₀ valueagainst recombinant human PDE5 of less than about 1 μM, and often lessthan about 0.05 μM. To achieve the full advantage of the invention, apresent PDE5 inhibitor has an IC₅₀ of about 0.1 nM to about 15 μM.

The production of recombinant human PDEs and the IC₅₀ determinations canbe accomplished by well-known methods in the art. Exemplary methods aredescribed as follows:

Expression of Human PDEs

Expression in Saccharomyces cerevisiae (Yeast)

Recombinant production of human PDE1B, PDE2, PDE4A, PDE4B, PDE4C, PDE4D,PDE5, and PDE7 was carried out similarly to that described in Example 7of U.S. Pat. No. 5,702,936, incorporated herein by reference, exceptthat the yeast transformation vector employed, which is derived from thebasic ADH2 plasmid described in Price et al., Methods in Enzymology,185, pp. 308-318 (1990), incorporated yeast ADH2 promoter and terminatorsequences and the Saccharomyces cerevisiae host was theprotease-deficient strain BJ2-54 deposited on Aug. 31, 1998 with theAmerican Type Culture Collection, Manassas, Va., under accession numberATCC 74465. Transformed host cells were grown in 2× SC-leu medium, pH6.2, with trace metals, and vitamins. After 24 hours, YEPmedium-containing glycerol was added to a final concentration of 2×YET/3% glycerol. Approximately 24 hr later, cells were harvested,washed, and stored at −70° C.

Human Phosphodiesterase Preparations

Phosphodiesterase Activity Determinations

Phosphodiesterase activity of the preparations was determined asfollows. PDE assays utilizing a charcoal separation technique wereperformed essentially as described in Loughney et al. (1996). In thisassay, PDE activity converts [32P]cAMP or [32P]cGMP to the corresponding[32P]5′-AMP or [32P]5′-GMP in proportion to the amount of PDE activitypresent. The [32P]5′-AMP or [32P]5′-GMP then was quantitativelyconverted to free [32P]phosphate and unlabeled adenosine or guanosine bythe action of snake venom 5′-nucleotidase. Hence, the amount of[32P]phosphate liberated is proportional to enzyme activity. The assaywas performed at 30° C. in a 100 μL reaction mixture containing (finalconcentrations) 40 mM Tris HCl (pH 8.0), 1 μM ZnSO₄, 5 mM MgCl₂, and 0.1mg/mL bovine serum albumin (BSA). PDE enzyme was present in quantitiesthat yield <30% total hydrolysis of substrate (linear assay conditions).The assay was initiated by addition of substrate (1 mM [32P]cAMP orcGMP), and the mixture was incubated for 12 minutes. Seventy-five (75)μg of Crotalus atrox venom then was added, and the incubation wascontinued for 3 minutes (15 minutes total). The reaction was stopped byaddition of 200 μL of activated charcoal (25 mg/mL suspension in 0.1 MNaH₂PO₄, pH 4). After centrifugation (750×g for 3 minutes) to sedimentthe charcoal, a sample of the supernatant was taken for radioactivitydetermination in a scintillation counter and the PDE activity wascalculated.

Purification of PDE5 from S. cerevisiae

Cell pellets (29 g) were thawed on ice with an equal volume of LysisBuffer (25 mM Tris HCl, pH 8, 5 mM MgCl₂, 0.25 mM DTT, 1 mM benzamidine,and 10 μM ZnSO₄). Cells were lysed in a Microfluidizer® (MicrofluidicsCorp.) using nitrogen at 20,000 psi. The lysate was centrifuged andfiltered through 0.45 μm disposable filters. The filtrate was applied toa 150 mL column of Q SEPHAROSE® Fast-Flow (Pharmacia). The column waswashed with 1.5 volumes of Buffer A (20 mM Bis-Tris Propane, pH 6.8, 1mM MgCl₂, 0.25 mM DTT, 10 μM ZnSO₄) and eluted with a step gradient of125 mM NaCl in Buffer A followed by a linear gradient of 125-1000 mMNaCl in Buffer A. Active fractions from the linear gradient were appliedto a 180 mL hydroxyapatite column in Buffer B (20 mM Bis-Tris Propane(pH 6.8), 1 mM MgCl₂, 0.25 mM DTT, 10 μM ZnSO₄, and 250 mM KCl). Afterloading, the column was washed with 2 volumes of Buffer B and elutedwith a linear gradient of 0-125 mM potassium phosphate in Buffer B.Active fractions were pooled, precipitated with 60% ammonium sulfate,and resuspended in Buffer C (20 mM Bis-Tris Propane, pH 6.8, 125 mMNaCl, 0.5 mM DTT, and 10 μM ZnSO₄). The pool was applied to a 140 mLcolumn of SEPHACRYL® S-300 HR and eluted with Buffer C. Active fractionswere diluted to 50% glycerol and stored at −20° C.

The resultant preparations were about 85% pure by SDS-PAGE. Thesepreparations had specific activities of about 3 μmol cGMP hydrolyzed perminute per milligram protein.

Inhibitory Effect on cGMP-PDE

cGMP-PDE activity of compounds of the present invention was measuredusing a one-step assay adapted from Wells et al., Biochim. Biophys.Acta, 384, 430 (1975). The reaction medium contained 50 mM Tris-HCl, pH7.5, 5 mM magnesium acetate, 250 μg/ml 5′-Nucleotidase, 1 mM EGTA, and0.15 μM 8-[H³]-cGMP. Unless otherwise indicated, the enzyme used was ahuman recombinant PDE5 (ICOS Corp., Bothell, Wash.).

Compounds of the invention were dissolved in DMSO finally present at 2%in the assay. The incubation time was 30 minutes during which the totalsubstrate conversion did not exceed 30%.

The IC₅₀ values for the compounds examined were determined fromconcentration-response curves typically using concentrations rangingfrom 10 nM to 10 μM. Tests against other PDE enzymes using standardmethodology showed that compounds of the invention are selective for thecGMP-specific PDE enzyme.

Biological Data

The compounds according to the present invention were typically found toexhibit an IC₅₀ value of less than 500 nM. An in vitro test usingExample 1, a representative compound of the invention, gave IC₅₀ versusPDE5 of 2.3 nM. Examples 4, 5(a), and 5(b) demonstrated an IC₅₀ versusPDE5 of 555 nM, 338 nM, and 125 nM, respectively.

Obviously, many modifications and variations of the invention ashereinbefore set forth can be made without departing from the spirit andscope thereof, and, therefore, only such limitations should be imposedas are indicated by the appended claims.

1. A compound having a formula

wherein R⁰, independently, is selected from the group consisting of haloand C₁₋₆alkyl; R¹ is selected from the group consisting of hydrogen,C₁₋₆alkyl, C₂₋₆alkenyl, C₂₋₆alkynyl, halo-C₁₋₆alkyl, C₃₋₈cycloalkyl,C₃₋₈cycloalkylC₁₋₃alkyl, and arylC₁₋₃alkyl; R² is selected from thegroup consisting of an optionally substituted monocyclic aromatic ringselected from the group consisting of benzene, thiophene, furan, andpyridine, and an optionally substituted bicyclic ring

wherein the fused ring A is a 5- or 6-membered ring, saturated orpartially or fully unsaturated, and comprises carbon atoms andoptionally one or two heteroatoms selected from oxygen, sulfur, andnitrogen, said optional substituents selected from the group consistingof halo, C₁₋₃alkyl, OR^(a), CO₂R^(a), halomethyl, halomethoxy, cyano,nitro, and N(R^(a))²; R³ is hydrogen or C₁₋₃alkyl; rings B and C form anoptionally substituted fused ring structure selected from the groupconsisting of

R^(a) is selected from the group consisting of hydrogen, C₁₋₆alkyl,C₃₋₈cycloalkyl, aryl, arylC₁₋₃alkyl, C₁₋₃alkylenearyl; q is 0, 1, 2, 3,or 4; or a pharmaceutically acceptable salt or hydrate thereof.
 2. Thecompound of claim 1 represented by the formula

or a pharmaceutically acceptable salt or hydrate thereof.
 3. Thecompound of claim 1 wherein R¹ is methyl.
 4. The compound of claim 1wherein R³ is hydrogen.
 5. The compound of claim 1 wherein rings B and Cform an optionally substituted fused ring structure selected from thegroup consisting of


6. The compound of claim 1 wherein R² is an optionally substitutedbicyclic ring selected from the group consisting of naphthalene, indene,benzoxazole, benzothiazole, benzisoxazole, benzimidazole, quinoline,indole, benzothiophene, and benzofuran.
 7. The compound of claim 1wherein R² is

wherein n is an integer 1 or 2, and X, independently, are C(R^(a))₂, O,S, or NR^(a).
 8. The compound of claim 1 wherein R², substituted orunsubstituted, is selected from the group consisting of


9. The compound of claim 8 wherein R² is substituted with a substituentselected from the group consisting of halogen, C₁₋₃alkyl, OR^(a),CO₂R^(a), halomethyl, halomethoxy, cyano, nitro, and N(R^(a))₂.
 10. Acompound selected from the group consisting of

or a pharmaceutically acceptable salt or hydrate thereof.
 11. Apharmaceutical composition comprising a compound of claim 1, togetherwith a pharmaceutically acceptable diluent or carrier.
 12. A method oftreating a male or female animal for male erectile dysfunction or femalearousal disorder comprising administering to said animal an effectiveamount of a pharmaceutical composition comprising a compound of claim 1,together with a pharmaceutically acceptable diluent or carrier.
 13. Themethod of claim 12 wherein the pharmaceutical composition isadministered orally.
 14. A compound having a formula

wherein R⁰, independently, is selected from the group consisting of haloand C₁₋₆alkyl; R¹ is selected from the group consisting of hydrogen,C₁₋₆alkyl, C₂₋₆alkenyl, C₂₋₆alkynyl, halo-C₁₋₆alkyl, C₃₋₈cycloalkyl,C₃₋₈cycloalkylC₁₋₃alkyl, and arylC₁₋₃alkyl; R² is selected from thegroup consisting of an optionally substituted monocyclic aromatic ringselected from the group consisting of benzene, thiophene, furan, andpyridine, and an optionally substituted bicyclic ring

wherein the fused ring A is a 5- or 6-membered ring, saturated orpartially or fully unsaturated, and comprises carbon atoms andoptionally one or two heteroatoms selected from oxygen, sulfur, andnitrogen, said optional substituents selected from the group consistingof halo, C₁₋₃alkyl, OR^(a), CO₂R^(a), halomethyl, halomethoxy, cyano,nitro, and N(R^(a))²; R³ is hydrogen or C₁₋₃alkyl; R^(a) is selectedfrom the group consisting of hydrogen, C₁₋₆alkyl, C₃₋₈cycloalkyl, aryl,arylC₁₋₃alkyl, C₁₋₃alkylenearyl. q is 0, 1, 2, 3, or 4; or apharmaceutically acceptable salt or hydrate thereof.